Dual specificity antibodies to pd-l1 and pd-l2 and methods of use therefor

ABSTRACT

The present disclosure is directed to dual specificity antibodies which may bind to PD-L1 or PD-L2 and methods of using such antibodies to treat cancers, such as those that express or overexpress PD-L1 or PD-L2.

PRIORITY CLAIM

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/647,562, filed Mar. 23, 2018, the entirecontents of which are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTFC.P1342WO.txt”, which is 22 KB (as measured in Microsoft Windows®)and was created on Mar. 13, 2019, is filed herewith by electronicsubmission and is incorporated by reference herein.

BACKGROUND 1. Field

The present disclosure relates generally to the field of medicine,oncology, and immunology. More particularly, it concerns humanantibodies binding to PD-L2 and their use in cancer therapies.

2. Description of Related Art

Blockade of the interaction of the T cell co-inhibitory receptor PD-1with its ligand PD-L1 has become a pillar of modern oncology nowavailable even in the first-line setting for subsets of melanoma andlung cancer patients (Boussiotis, 2016). Numerous antibodies targetingPD-1 or PD-L1 are currently FDA approved or in clinical trials; however,no agents targeting the second PD-1 ligand, PD-L2, are under clinicalinvestigation. PD-L2 binds PD-1 with an approximately 3-fold higheraffinity than does PD-L1, and, like PD-L1, sends an inhibitory signalwhich attenuates T cell function (Cheng el al., 2013; Latchman et al.,2001; Lee et al., 2016; Li et al., 2017; Youngnak et al., 2003).Historically, PD-L2 was largely considered to be an inducibleco-inhibitory molecule with expression limited to the tumor stroma;however, improved detection reagents for PD-L2 have revealed widespreadPD-L2 expression both in the tumor microenvironment and on tumor cellsthemselves (Baptista et al., 2016; Danilova et al., 2016; Derks et al.,2015; Dong et al., 2016; Howitt et al., 2016; Kim et al., 2015; Kim etal., 2015; Nomi et al., 2007; Obeid et al., 2016; Ohigashi et al., 2005;Roemer et al., 2016; Shi et al., 2014; Shin et al., 2015; Xu et al.,2016). Recently, PD-L2 was shown to be an independent predictor ofresponse to the PD-1 antibody pembrolizumab across multiple cancers(Yearley et al., 2017).

First described in the vast majority of classical Hodgkin's Lymphoma(cHL), amplification of chromosomal region 9p24.1 leads to directupregulation of PD-L1 and PD-L2 (which reside therein), as well asindirect induction via enhanced JAK2 activity (Roemer et al., 2016; Shiet al., 2014; Green et al., 2010; Van Roosbroeck et al., 2016). Inaddition to cHL, this genetic driver of high PD-L1/PD-L2 co-expressionis also found in the majority of Primary Mediastinal Large B-CellLymphoma (PMBL), T cell lymphoma, and a variety of histiocytic anddendritic cell malignancies. Not surprisingly, many of these cancershave been shown to respond to PD-1 blockade. More recently, 9p24.1amplification has been demonstrated in solid tumors such astriple-negative breast cancer (TNBC) (Howitt et al, 2016; Barrett et al,2015). Relatively high co-expression of PD-L1 and PD-L2 has also beenobserved in a number of other cancers such as gastric carcinoma,melanoma, squamous carcinomas of the lung, head and neck, cervix andvulva, bladder cancer, and hepatocellular carcinoma among others(Baptista et al., 2016; Danilova et al., 2016; Derks et al., 2015; Donget al., 2016; Howitt et al., 2016; Kim et al, 2015; Nomi et al., 2007;Obeid et al., 2016; Xu et al., 2016; Yearley et al., 2017; VanRoosbroeck et al., 2016; Barrett et al., 2015; Shin et al., 2016; Inoueet al., 2016; Wang et al., 2011). In addition to expression by thesetumors themselves, stromal and endothelial expression of PD-L2 has alsobeen documented for many of these tumors (Yearley et al., 2017). Thesefindings suggest limitations to the therapeutic potential of PD-L1blockade in these cancers.

The PD-1 co-inhibitory receptor is expressed primarily by activated Tcells and NK cells and can thus best be targeted by antibodies whichbind it and prevent engagement by PD-ligands. PD-L1, in contrast, isexpressed by tumor cells and suppressive stromal populations and can betargeted with antibodies capable of cytotoxic effector function. Whilethe theoretical advantages of these antibody-dependent cellularcytotoxicity (AMC) capable PD-L1 antibodies can be demonstrated invitro, no patient data exists demonstrating actual effector function inpatients or improved outcome relative to purely blocking variants(Boyerinas et al., 2015).

PD-L1 and PD-L2 share only approximately 40% identity, as each binds anadditional receptor distinct from PD-1 (Latchman et al., 2001). PD-L1also binds B7-1 in an additional negative T cell regulatory interaction(Butte et al., 2007; Butte et al., 2008). In mice, PD-L2 can bind toRGMb on either myeloid cells or T cells and regulate tolerance toinhaled antigens (Xiao et al., 2014; Nie et al., 2017). The role ofPD-L2 binding to RGMb in tumors remains to be described, as does therelevance of this interaction in humans. It could prove extremelyadvantageous from a therapeutic standpoint to have bispecific antibodiesto PD-L1 and PD-L2.

SUMMARY

Thus, in accordance with the present disclosure, there is provided adual specificity antibody or antibody fragment where one light and heavychain pair binds selectively to PD-L1, and another light and heavy chainpair binds selectively to PD-L2, comprising a first set of clone pairedheavy and light CDR sequences selected from SEQ ID NOS: 19-24 or 25-30,and a second set of clone paired heavy and light CDR sequences selectedfrom SEQ ID NOS: 31-36 or 37-42. The antibody or antibody fragment maybe encoded by a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10, may be encoded by two sets of paired heavy andlight chain variable sequences having at least 70%, 80%, or 90% identityto a first set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 3-4 or 5-6, and a second set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 7-8or 9-10, or may be encoded by two sets of paired heavy and light chainvariable sequences having at least 95% identity to a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 3-4 or 5-6, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 7-8 or 9-10. The antibodyor antibody fragment may comprise a first set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 11-12 or 13-14,and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 15-16 or 17-18, may comprise twosets of paired heavy and light chain variable sequences having at least70%, 80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18, or may comprise two sets ofpaired heavy and light chain variable sequences having 95% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18.

There is also provided a method of treating cancer in a subjectcomprising contacting a PD-L1 or PD-L2 positive cancer cell in a subjectwith an antibody as described above. The PD-L1 or PD-L2 positive cancercell may be a solid tumor cell, such as a lung cancer cell, brain cancercell, head & neck cancer cell, breast cancer cell, skin cancer cell,liver cancer cell, pancreatic cancer cell, stomach cancer cell, coloncancer cell, rectal cancer cell, uterine cancer cell, cervical cancercell, ovarian cancer cell, testicular cancer cell, skin cancer cell,esophageal cancer cell, a lymphoma cell, a renal cell carcinoma cell, ormay be a leukemia or myeloma such as acute myeloid leukemia, chronicmyelogenous leukemia or multiple myeloma.

The method may further comprise contacting the PD-L1 or PD-L2 positivecancer cell with a second anti-cancer agent or treatment, such aschemotherapy, radiotherapy, immunotherapy, hormonal therapy, or toxintherapy. The second anti-cancer agent or treatment may inhibit anintracellular PD-L1 or PD-L2 function. The second anti-cancer agent ortreatment may be given at the same time as the first agent, or givenbefore and/or after the agent. The PD-L1 or PD-L2 positive cancer cellmay be a metastatic cancer cell, a multiply drug resistant cancer cellor a recurrent cancer cell.

There is also provided a method of treating immune suppression in atumor microenvironment in a subject having cancer comprising deliveringto the subject an antibody or antibody fragment as described above.

The antibody may be a single chain antibody, a single domain antibody, achimeric antibody, or a Fab fragment. The antibody may be a humanantibody, murine antibody, an IgG, a humanized antibody or a humanizedIgG. The antibody or antibody fragment may further comprise a label,such as a peptide tag, an enzyme, a magnetic particle, a chromophore, afluorescent molecule, a chemiluminescent molecule, or a dye. Theantibody or antibody fragment may further comprise an antitumor druglinked thereto, such as linked to the antibody or antibody fragmentthrough a photolabile linker or an enzymatically-cleaved linker. Theantitumor drug may be a toxin, a radioisotope, a cytokine or an enzyme.The antibody or antibody fragment may be conjugated to a nanoparticle ora liposome.

In another embodiment, there is provided a method of treating a cancerin a subject comprising an antibody or antibody fragment comprising afirst set of clone paired heavy and light CDR sequences selected fromSEQ ID NOS: 19-24 or 25-30, and a second set of clone paired heavy andlight CDR sequences selected from SEQ ID NOS: 31-36 or 37-42. Theantibody fragment may be a recombinant scFv (single chain fragmentvariable) antibody, Fab fragment, F(ab′)₂ fragment, or Fv fragment. Theantibody may be an IgG. The antibody may be a chimeric antibody.Delivering may comprise antibody or antibody fragment administration, orgenetic delivery with an RNA or DNA sequence or vector encoding theantibody or antibody fragment.

The antibody or antibody fragment may be encoded by a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 3-4 or 5-6, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 7-8 or 9-10, may be encodedby two sets of paired heavy and light chain variable sequences having atleast 70%, 80%, or 90% identity to a first set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, anda second set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, and may be encoded by two sets ofpaired heavy and light chain variable sequences having at least 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10. The antibody or antibody fragment may comprisecomprises a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 11-12 or 13-14, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 15-16 or 17-18, may comprise two sets of paired heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, or may comprise two sets of paired heavy and lightchain variable sequences having at least 95% identity to a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18.

Also provided is a monoclonal antibody, wherein the antibody or antibodyfragment is characterized by a first set of clone paired heavy and lightCDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and a second setof clone paired heavy and light CDR sequences selected from SEQ ID NOS:31-36 or 37-42. The antibody fragment may be a recombinant scFv (singlechain fragment variable) antibody, Fab fragment, F(ab′)₂ fragment, or Fvfragment. The antibody may be a chimeric antibody, or an IgG.

The antibody or antibody fragment may be encoded by a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 3-4 or 5-6, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 7-8 or 9-10, may be encodedby two sets of paired heavy and light chain variable sequences having atleast 70%, 80%, or 90% identity to a first set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, anda second set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, and may be encoded by two sets ofpaired heavy and light chain variable sequences having at least 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10. The antibody or antibody fragment may comprise afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, may comprise two sets of paired heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, or may comprise two sets of paired heavy and lightchain variable sequences having 95% identity to a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18.

In yet another embodiment, there is provided a hybridoma or engineeredcell encoding an antibody or antibody fragment wherein the antibody orantibody fragment is characterized by a first set of clone paired heavyand light CDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and asecond set of clone paired heavy and light CDR sequences selected fromSEQ ID NOS: 31-36 or 37-42. The antibody fragment may be a recombinantscFv (single chain fragment variable) antibody, Fab fragment, F(ab′)₂fragment, or Fv fragment. The antibody may be a chimeric antibody or anIgG.

The antibody or antibody fragment may be encoded by a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 3-4 or 5-6, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 7-8 or 9-10, may be encodedby a first set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 3-4 or 5-6, and a second set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 7-8or 9-10, may be encoded by two sets of paired heavy and light chainvariable sequences having at least 70%, 80%, or 90% identity to a firstset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 3-4 or 5-6, and a second set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 7-8 or 9-10,and may be encoded by two sets of paired heavy and light chain variablesequences having at least 95% identity to a first set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 3-4or 5-6, and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10. The antibody orantibody fragment may comprise a first set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 11-12 or 13-14,and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 15-16 or 17-18, may comprise twosets of paired heavy and light chain variable sequences having at leak70%, 80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18, or may comprise two sets ofpaired heavy and light chain variable sequences having 95% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18.

A further embodiment comprises a cancer vaccine comprising one or moreantibodies or antibody fragments characterized a first set of clonepaired heavy and light CDR sequences selected from SEQ ID NOS: 19-24 or25-30, and a second set of clone paired heavy and light CDR sequencesselected from SEQ ID NOS: 31-36 or 37-42. At least one antibody fragmentmay be a recombinant ScFv (single chain fragment variable) antibody, Fabfragment, F(ab′)₂ fragment, or Fv fragment. At least one of antibody maybe a chimeric antibody, or an IgG. At least one antibody or antibodyfragment may be encoded by a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, may be encoded by two sets ofpaired heavy and light chain variable sequences having at least 70%,80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, and may be encoded encoded by twosets of paired heavy and light chain variable sequences having at least95% identity to a first set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 3-4 or 5-6, and a secondset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 7-8 or 9-10. At least one antibody or antibody fragmentmay comprise a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 11-12 or 13-14, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 15-16 or 17-18, may comprise two sets of paired heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to a.first set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, or may comprise two sets of paired heavy and lightchain variable sequences having 95% identity to a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18.

In another embodiment there is provided a method of detecting PD-L1 orPD-L2 expressing cells in a subject comprising contacting a sample fromsaid subject with an antibody or antibody fragment having a first set ofclone paired heavy and light CDR sequences selected from SEQ ID NOS:19-24 or 25-30, and a second set of clone paired heavy and light CDRsequences selected from SEQ ID NOS: 31-36 or 37-42, and detecting aPD-L1 or PD-L2 expressing cell in said sample by binding said antibodyor antibody fragment to a cell in said sample. The sample may be a bodyfluid or a tissue sample. The cell may be a cancer cell, such as alymphoma cell, breast cancer cell, or renal cell carcinoma cell. Thecell may be a cell associated with immune suppression. The cellassociated with immune suppression may be a non-cancerous cell in atumor microenvironment, such as a stromal cell or endothelial cell.Detection may comprise ELISA, RIA, or Western blot. The method mayfurther comprise performing the method a second time and determining achange in antigen levels as compared to the first assay. The antibody orantibody fragment may be encoded by a first set of clone paired heavyand light chain variable sequences selected from SEQ ID NOS: 3-4 or 5-6,and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10, may be encoded by twosets of paired heavy and light chain variable sequences having at least70%, 80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, and may be encoded by two sets ofpaired heavy and light chain variable sequences having at least 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10. The antibody or antibody fragment may comprise afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, may comprise two sets of paired heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ_(.) ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, or may comprise two sets of paired heavy and lightchain variable sequences having 95% identity to a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18.

Also provided is a method of treating immune suppression in a tumormicroenvironment in a subject having cancer comprising delivering tosaid subject an antibody or antibody fragment having a first set ofclone paired heavy and light CDR sequences selected from SEQ ID NOS:19-24 or 25-30, and a second set of clone paired heavy and light CDRsequences selected from SEQ ID NOS: 31-36 or 37-42. The antibody orantibody fragment may be encoded by a first set of clone paired heavyand light chain variable sequences selected from SEQ ID NOS: 3-4 or 5-6,and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10, may be encoded by twosets of paired heavy and light chain variable sequences having at least70%, 80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10, and may be encoded by two sets ofpaired heavy and light chain variable sequences having at least 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10. The antibody or antibody fragment may comprise afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, may comprise two sets of paired heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18, or may comprise two sets of paired heavy and lightchain variable sequences having 95% identity to a first set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Other objects, features and advantages of the present disclosurewill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis preferably below 0.01%. Most preferred is a composition in which noamount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Selection of Multiple Families of PD-L1/PD-L2 Antibodies Sharinga Common Light Chain. Pictured is a diagram flow chart showing thatPD-L1 and PD-L2 heavy chains from four rounds of selection were pairedwith 9 distinct common light chain libraries (cLC) and selected forPD-L1 and PD-L2 binding antibodies. These antibodies were then subjectedto affinity maturation using CDR-H1 and CDR-H2 libraries to yield thefinal clones.

FIG. 2—DiPDL Antibodies are Equivalent to PD-1 Antibodies inPD-1:PD-L1/PD-L2 Blockade Assay. Candidate PD-L1, PD-L2, or assembledDiPDL mAbs were assayed using the Promega® PD-L1, PD-L2, or PD-L1/PD-L2:PD-1 blockade system as indicated. Varying concentrations of antibodywere added to CHO-PD-L1 and/or PD-L2 cells capable of stimulating JurkatT cells. Jurkat T cells which produce firefly Luciferase in response toactivation were incubated with antibody and CHO cells for 6 hours andthen the results were read out on a luminometer using the Bio-Glo™ assaykit (Promega®).

FIG. 3—DiPDL Antibodies Trigger IFN-γ Release Equivalently to Keytrudain Human Mixed Lymphocyte Reactions. Varying concentrations of cLCPD-L1, PD-L2 or DiPDL antibodies (human IgG1) were added to inducedhuman dendritic cells from a single selected donor. T cells from aseparate donor were added at a ratio of 10 to 1 and either IL-2 and/orIFN-γ production was detected by ELISA.

FIG. 4—DiPDL Antibodies Mediate Efficient ADCC Against Human PD-L1/L2⁺Lymphoma. Varying concentrations of PD-L1, PD-L2, or DiPDL, antibodies(human IgG1) were added with in vitro expanded murine NK cells or humanPBMC to Calcein (ThermoFisher®)-labelled U2940 PMBL cells at an effectorto target ratio of 15 to 1. Percent specific lysis was calculated as thedifference between experimental release and spontaneous release ofCalcein as measured on a fluorescent plate reader.

FIG. 5—DiPDL Antibodies with ADCC are Active Against Etonian U2940Lymphoma in vivo. U2940 PBML xenograft tumors were established in SCIDmice, allowed to reach 150 mm³ and then treated with the indicatedantibodies intraperitoneally 2×/week at 10 mg/kg for 3 weeks. Data shownis from a single experiment with 9 mice per group.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors have generated monoclonal antibodies with bindingspecificity for human PD-L2 protein. As these antibodies have beendemonstrated to bind to PD-L2, they present an opportunity to block thebinding of PD-L2 to PD-1. They can also be used to deliver therapeuticpayloads to PD-L2 expressing cancer cells. These and other aspects ofthe disclosure are described in even greater detail below.

I. PD-L1

A. Structure

Programmed death-ligand 1 (PD-L1) is a protein encoded by the CD274gene. PD-L1 is a 40 kDa type 1 transmembrane protein which may play amajor role in immune suppression during a variety of events such as,pregnancy, tissue allografts, autoimmune disease, cancer and otherdisease states. The human PD-L1 protein is encoded by the amino acidsequence shown below:

(SEQ ID NO: 1) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

B. Function

PD-L1 is a ligand to its receptor, PD-1. PD-1 may be found on activatedT cells, B cells, and myeloid cells. Binding of PD-L1 to PD-1 modulatesT cell and B cell activation or inhibition. transmits an inhibitorsignal that reduces proliferation of antigen specific CD8+ T cells andCD4+ helper T-cells. Binding of PD-L1 to PD-1 also induces apoptosis.This reduction of CD8+ T cells and CD4+ helper T-cells has been thoughtto help PD-L1 expressing cancer cells evade anti-tumor immunity (Dong etal, Nat. Medicine, 2002). Upregulation of PD-L1 has been associated withevasion of the host immune system, and is thought to be a cause ofincreased tumor aggressiveness (Thompson et al., 2004). The role ofPD-L1 in evasion of anti-tumor immunity makes it an attractive targetfor therapeutic intervention.

II. PD-L2

A. Structure

Programmed death-ligand 2 (PD-L2) is a protein encoded by the CD273gene. PD-L2 is a 31 kDa protein which may play a major role in immunesuppression during a variety of events such as, pregnancy, tissueallografts, autoimmune disease, cancer and other disease states. Thehuman PD-L2 protein is encoded by the amino acid sequence shown below:

(SEQ ID NO: 2) MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECKFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI

PD-L2 is initially produced with a signal peptide corresponding to aminoacids 1-19 of SEQ ID NO. 2, which is subsequently removed to yield themature protein. The mature PD-L2 protein, corresponding to amino acids20-273 of SEQ ID NO. 2, is comprised of an Ig-like V-domain, an Ig-likeC2-type domain, transmembrane domain, and a cytoplasmic tail.

B. Function

PD-L2 is a ligand to its receptor, PD-1. PD-1 may be found on activatedT cells, B cells, and myeloid cells. Binding of PD-L2 to PD-1 begins animmunological cascade which impairs proliferation, cytokine production,cytolytic function and survival of the T cell. PD-1 transmits aninhibitor signal that reduces proliferation of antigen specific CD8+ Tcells and CD4+ helper T-cells. PD-L2 has also been shown to be anindependent predictor of response to the PD-1 antibody pembrolizumabacross multiple cancers (Yearley et al., 2017).

III. MONOCLONAL ANTIBODIES AND PRODUCTION THEREOF

A. General Methods

Antibodies having dual specificity which bind to both PD-L1 and PD-L2(DiPDL) may be produced by standard methods as are well known in the art(see, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; U.S. Pat. No. 4,196,265). The methods for generatingmonoclonal antibodies (mAbs) generally begin along the same lines asthose for preparing polyclonal antibodies. The first step for both thesemethods is immunization of an appropriate host or identification ofsubjects who are immune due to prior natural infection. As is well knownin the art, a given composition for immunization may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine. As also is well known in the art, theimmunogenicity of a particular immunogen composition can be enhanced bythe use of non-specific stimulators of the immune response, known asadjuvants. Exemplary and preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, also may be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate monoclonal antibodies.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens or lymph nodes, or from circulating blood. Theantibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized or human or human/mousechimeric cells. Myeloma cell lines suited for use in hybridoma-producingfusion procedures preferably are non-antibody-producing, have highfusion efficiency, and enzyme deficiencies that render then incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 413210; and U-266, GM1500-GRG2, LICR-LON-HMy2and UC729-6 are all useful in connection with human cell fusions. Oneparticular murine myeloma. cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline. More recently, additional fusion partner lines for use with humanB cells have been described, including KR12 (ATCC CRL-8658; K6H6/B5(ATCC CRL-1823 SHM-D33 (ATCC CRL-1668) and HMMA2.5 (Posner et at.,1987). The antibodies in this disclosure were generated using theSP2/0/mIL-6 cell line, an IL-6 secreting derivative of the SP2/0 line.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods also is appropriate (Goding, pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, infusedcells (particularly the infused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.Ouabain is added if the B cell source is an Epstein Barr virus (EBV)transformed human B cell line, in order to eliminate EBV transformedlines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cellscapable of operating nucleotide salvage pathways are able to survive inHAT medium. The myeloma cells are defective in key enzymes of thesalvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT),and they cannot survive. The B cells can operate this pathway, but theyhave a limited life span in culture and generally die within about twoweeks. Therefore, the only cells that can survive in the selective mediaare those hybrids formed from myeloma and B cells. When the source of Bcells used for fusion is a line of EBV-transformed B cells, as here,ouabain is also used for drug selection of hybrids as EBV-transformed Bcells are susceptible to drug killing, whereas the myeloma partner usedis chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microliter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays dot immunobindingassays, and the like.

The selected hybridomas are then serially diluted or single-cell sortedby flow cytometric sorting and cloned into individual antibody-producingcell lines, which clones can then be propagated indefinitely to providemAbs. The cell lines may be exploited for MAb production in two basicways. A sample of the hybridoma can be injected (often into theperitoneal cavity) into an animal (e.g., a mouse). Optionally, theanimals are primed with a hydrocarbon, especially oils such as pristane(tetramethylpentadecane) prior to injection. When human hybridomas areused in this way, it is optimal to inject immunocompromised mice, suchas SCID mice, to prevent tumor rejection. The injected animal developstumors secreting the specific monoclonal antibody produced by the fusedcell hybrid. The body fluids of the animal, such as serum or ascitesfluid, can then be tapped to provide mAbs in high concentration. Theindividual cell lines could also be cultured in vitro, where the mAbsare naturally secreted into the culture medium from which they can bereadily obtained in high concentrations. Alternatively, human hybridomacells lines can be used in vitro to produce immunoglobulins in cellsupernatant. The cell lines can be adapted for growth in serum-freemedium to optimize the ability to recover human monoclonalimmunoglobulins of high purity.

Monoclonal antibodies produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as FPLC or affinity chromatography. Fragments of themonoclonal antibodies of the disclosure can be obtained from thepurified monoclonal antibodies by methods which include digestion withenzymes, such as pepsin or papain, and/or by cleavage of disulfide bondsby chemical reduction. Alternatively, monoclonal antibody fragmentsencompassed by the present disclosure can be synthesized using anautomated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. For this, RNA can be isolated from thehybridoma line and the antibody genes obtained by RT-PCR and cloned intoan immunoglobulin expression vector. Alternatively, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe cell lines and phagemids expressing appropriate antibodies areselected by panning using viral antigens. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by H and L chaincombination which further increases the chance of finding appropriateantibodies.

Yeast-based antibody presentation libraries may be designed rationally,and antibodies may be selected and/or isolated from such yeast-basedantibody presentation libraries, as disclosed in, for example,WO2012/009568; WO2009/036379; WO2010/105256; WO2003/074679; U.S. Pat.Nos. 8,691,730; and 9,354,228 The antibodies may then be expressed asfull length IgGs from the desired cell type and purified.

Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present disclosure includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobulin preparations; and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in thefirst instance, by their binding specificity, i.e., dual specificity forbinding to PD-L1 or PD-L2. Those of skill in the art, by assessing thebinding specificity/affinity of a given antibody using techniques wellknown to those of skill in the art, can determine whether suchantibodies fall within the scope of the instant claims. In one aspect,there are provided monoclonal antibodies having clone-paired CDR's fromthe heavy and light chains as illustrated in Tables 3 and 4,respectively. Such antibodies may be produced by the clones discussedbelow in the Examples section using methods described herein.

In a second aspect, the antibodies may be defined by their variablesequence, which include additional “framework” regions. These areprovided in Tables 1 and 2 that encode or represent full variableregions. Furthermore, the antibodies sequences may vary from thesesequences, optionally using methods discussed in greater detail below.For example, nucleic acid sequences may vary from those set out above inthat (a) the variable regions may be segregated away from the constantdomains of the heavy and light chains, (b) the nucleic acids may varyfrom those set out above while not affecting the residues encodedthereby, (c) the nucleic acids may vary from those set out above by agiven percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary fromthose set out above by virtue of the ability to hybridize under highstringency conditions, as exemplified by low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C., (e) the aminoacids may vary from those set out above by a given percentage, e.g.,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology,or (f) the amino acids may vary from those set out above by permittingconservative substitutions (discussed below). Each of the foregoingapplies to the nucleic acid sequences set forth as Table 1 and the aminoacid sequences of Table 2.

C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of theidentified antibodies for a variety of reasons, such as improvedexpression, improved cross-reactivity or diminished off-target binding.The following is a general discussion of relevant techniques forantibody engineering.

Hybridornas may be cultured, then cells lysed, and total RNA extracted.Random hexamers may be used with RT to generate cDNA copies of RNA, andthen PCR performed using a multiplex mixture of PCR primers expected toamplify all human variable gene sequences. PCR product can be clonedinto pGEM-T Easy vector, then sequenced by automated DNA sequencingusing standard vector primers. Assay of binding and neutralization maybe performed using antibodies collected from hybridoma supernatants andpurified by FPLC, using Protein G columns.

Recombinant full length IgG antibodies were generated by subcloningheavy and light chain TN DNAs from the cloning vector into an IgGplasmid vector, transfected into 293 Freestyle cells or CHO cells, andantibodies were collected an purified from the 293 or CHO cellsupernatant.

The rapid availability of antibody produced in the same host cell andcell culture process as the final cGMP manufacturing process has thepotential to reduce the duration of process development programs. Lonzahas developed a generic method using pooled transfectants grown in CDACFmedium, for the rapid production of small quantities (up to 50 g) ofantibodies in CHO cells. Although slightly slower than a true transientsystem, the advantages include a higher product concentration and use ofthe same host and process as the production cell line. Example of growthand productivity of GS-CHO pools, expressing a model antibody, in adisposable bioreactor: in a disposable bag bioreactor culture (5 Lworking volume) operated in fed-batch mode, a harvest antibodyconcentration of 2 g/L was achieved within 9 weeks of transfection.

Antibodies, and antibody libraries from which such antibodies may beselected and/or isolated, may be rationally designed and synthesized,such as by the Adimab® technology, as disclosed in, for example,WO2012/009568; WO2009/036379; WO2010/105256; WO2003/074679; U.S. Pat.Nos. 8,691,730; and 9,354,228. This method of synthesis antibodiesrequires that the nucleotide sequence coding for the desired or designedantibody be inserted into a vector for ectopic expression. Then thedesired antibodies may be expressed as full chain IgG molecules andpurified.

Antibody molecules will comprise fragments (such as F(ab′), F(ab′)₂)that are produced, for example, by the proteolytic cleavage of the mAbs,or single-chain immunoglobulins producible, for example, via recombinantmeans. Such antibody derivatives are monovalent. In one embodiment, suchfragments can be combined with one another, or with other antibodyfragments or receptor ligands to form “chimeric” binding molecules.Significantly, such chimeric molecules may contain substituents capableof binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosedantibodies, e.g., an antibody comprising the CDR sequences identical tothose in the disclosed antibodies (e.g., a chimeric, or CDR-graftedantibody). Alternatively, one may wish to make modifications, such asintroducing conservative changes into an antibody molecule. In makingsuch changes, the hydropathic index of amino acids may be considered.The importance of the hydropathic amino acid index in conferringinteractive biologic function on a protein is generally understood inthe art (Kyte and Doolittle, 1982). It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: basic amino acids: arginine (+3.0), lysine (+3.0), andhistidine (−0.5); acidic amino acids: aspartate (+3.0±1), glutamate(+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionicamino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), andthreonine (−0.4), sulfur containing amino acids: cysteine (−1.0) andmethionine (−1.3); hydrophobic, nonaromatic amino acids: valine (−1.5),leucine isoleucine (−1.8), proline (−0.5±1), alanine (−0.5), and glycine(0); hydrophobic, aromatic amino acids: tryptophan (−3.4), phenylalanine(−2.5), and tyrosine (−2.3).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity and produce a biologically orimmunologically modified protein. In such changes, the substitution ofamino acids whose hydrophilicity values are within ±2. is preferred,those that are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. Bymodifying the Fc region to have a different isotype, differentfunctionalities can be achieved. For example, changing to IgG₁ canincrease antibody dependent cell cytotoxicity, switching to class A canimprove tissue distribution, and switching to class M can improvevalency.

Modified antibodies may be made by any technique known to those of skillin the art, including expression through standard molecular biologicaltechniques, or the chemical synthesis of polypeptides. Methods forrecombinant expression are addressed elsewhere in this document.

D. Single Chain Antibodies

A Single Chain Variable Fragment (scFv) is a fusion of the variableregions of the heavy and light chains of immunoglobulins, linkedtogether with a short (usually serine, glycine) linker. This chimericmolecule retains the specificity of the original immunoglobulin, despiteremoval of the constant regions and the introduction of a linkerpeptide. This modification usually leaves the specificity unaltered.These molecules were created historically to facilitate phage displaywhere it is highly convenient to express the antigen binding domain as asingle peptide. Alternatively, scFv can be created directly fromsubcloned heavy and light chains derived from a hybridoma. Single chainvariable fragments lack the constant Fe region found in completeantibody molecules, and thus, the common binding sites (e.g., proteinA/G) used to purify antibodies. These fragments can often bepurified/immobilized using Protein L since Protein L interacts with thevariable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promotingamino acid residues such as Maine, serine and glycine. However, otherresidues can function as well. Tang et al. (1996) used phage display asa means of rapidly selecting tailored linkers for single-chainantibodies (scFvs) from protein linker libraries.: random linker librarywas constructed in which the genes for the heavy and light chainvariable domains were linked by a segment encoding an 18-amino acidpolypeptide of variable composition. The scFv repertoire (approx. 5×10⁶different members) was displayed on filamentous phage and subjected toaffinity selection with hapten. The population of selected variantsexhibited significant increases in binding activity but retainedconsiderable sequence diversity. Screening 1054 individual variantssubsequently yielded a catalytically active scFv that was producedefficiently in soluble form. Sequence analysis revealed a conservedproline in the linker two residues after the V_(H) C terminus and anabundance of arginines and prolines at other positions as the onlycommon features of the selected tethers.

The recombinant antibodies of the present disclosure may also involvesequences or moieties that permit dimerization or multimerization of thereceptors. Such sequences include those derived from IgA, which permitformation of multimers in conjunction with the J-chain. Anothermultimerization domain is the Gal4 dimerization domain. In otherembodiments, the chains may be modified with agents such asbiotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created byjoining receptor heavy and light chains using a non-peptide linker orchemical unit. Generally, the heavy and light chains will be produced indistinct cells, purified, and subsequently linked together in anappropriate fashion (i.e., the N-terminus of the heavy chain beingattached to the C-terminus of the light chain via an appropriatechemical bridge).

Cross-linking reagents are used to form molecular bridges that tiefunctional groups of two different molecules, e.g., a stabilizing andcoagulating agent. However, it is contemplated that dimers or multimersof the same analog or heteromeric complexes comprised of differentanalogs can be created. To link two different compounds in a step-wisemanner, hetero-bifunctional cross-linkers can be used that eliminateunwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo,preventing release of the targeting peptide prior to reaching the siteof action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amities (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4.680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Particular uses include adding a free amino or free sulfhydryl group toa protein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

E. Purification

In certain embodiments, the antibodies of the present disclosure may bepurified. The term “purified,” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein ispurified to any degree relative to its naturally-obtainable state. Apurified protein therefore also refers to a protein, free from theenvironment in which it may naturally occur. Where the term“substantially purified” is used, this designation will refer to acomposition in which the protein or peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the proteins in thecomposition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. Other methods for protein purification include,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; gel filtration, reversephase, hydroxylapatite and affinity chromatography; and combinations ofsuch and other techniques.

In purifying an antibody of the present disclosure, it may be desirableto express the polypeptide in a prokaryotic or eukaryotic expressionsystem and extract the protein using denaturing conditions. Thepolypeptide may be purified from other cellular components using anaffinity column, which binds to a tagged portion of the polypeptide. Asis generally known in the art, it is believed that the order ofconducting the various purification steps may be changed, or thatcertain steps may be omitted, and still result in a suitable method forthe preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e.,protein A) that bind the Fe portion of the antibody. Alternatively,antigens may be used to simultaneously purify and select appropriateantibodies. Such methods often utilize the selection agent bound to asupport, such as a column, filter or bead. The antibodies is bound to asupport, contaminants removed (e.g., washed away), and the antibodiesreleased by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. Another method forassessing the purity of a fraction is to calculate the specific activityof the fraction, to compare it to the specific activity of the initialextract, and to thus calculate the degree of purity. The actual unitsused to represent the amount of activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed protein or peptide exhibits adetectable activity.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

IV. PHARMACEUTICAL FORMULATIONS AND TREATMENT OF CANCER

A. Cancers

Cancer results from the outgrowth of a clonal population of cells fromtissue. The development of cancer, referred to as carcinogenesis, can bemodeled and characterized in a number of ways. An association betweenthe development of cancer and inflammation has long-been appreciated.The inflammatory response is involved in the host defense againstmicrobial infection, and also drives tissue repair and regeneration.Considerable evidence points to a connection between inflammation and arisk of developing cancer, i.e., chronic inflammation can lead todysplasia.

Cancer cells to which the methods of the present disclosure can beapplied include generally any cell that expresses PD-L1 or PD-L2, andmore particularly, that overexpresses PD-L1 or PD-L2. An appropriatecancer cell can be a breast cancer, lung cancer, colon cancer,pancreatic cancer, renal cancer, stomach cancer, liver cancer, bonecancer, hematological cancer (e.g., leukemia or lymphoma), neural tissuecancer, melanoma, ovarian cancer, testicular cancer, prostate cancer,cervical cancer, vaginal cancer, or bladder cancer cell. In addition,the methods of the disclosure can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. Cancers may also berecurrent, metastatic and/or multi-drug resistant, and the methods ofthe present disclosure may be particularly applied to such cancers so asto render them resectable, to prolong or re-induce remission, to inhibitangiogenesis, to prevent or limit metastasis, and/or to treat multi-drugresistant cancers. At a cellular level, this may translate into killingcancer cells, inhibiting cancer cell growth, or otherwise reversing orreducing the malignant phenotype of tumor cells.

B. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprisingdual specificity PD-L1 and PD-L2 antibodies (DiPDL). In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Other suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol and the like.

The compositions can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with anions suchas those derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with cations such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The antibodies of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by intradermal, subcutaneous, intramuscular, intraperitoneal orintravenous injection. Such compositions would normally be administeredas pharmaceutically acceptable compositions, described supra. Ofparticular interest is direct intratumoral administration, perfusion ofa tumor, or admininstration local or regional to a tumor, for example,in the local or regional vasculature or lymphatic system, or in aresected tumor bed.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

C. Combination Therapies

in the context of the present disclosure, it also is contemplated thatdual specificity antibodies to PD-L1 and PD-L2 described herein could beused similarly in conjunction with chemo- or radiotherapeuticintervention, or other treatments. It also may prove effective, inparticular, to combine dual specificity PD-L1 and PD-L2 antibodies withother therapies that target different aspects of PD-L1 or PD-L2function, such as peptides and small molecules that target the PD-L1 orPD-L2 cytoplasmic domain.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentdisclosure, one would generally contact a “target” cell with a dualspecificity PD-L1 and PD-L2. antibody according to the presentdisclosure and at least one other agent. These compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with the dualspecificity PD-L1 and PD-L2 antibody according to the present disclosureand the other agent(s) or factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the dual specificity PD-L1 andPD-L2 antibody according to the present disclosure and the otherincludes the other agent.

Alternatively, the DiPDL antibody therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and the dual specificity antibody toPD-L1 and PD-L2 are applied separately to the cell, one would generallyensure that a significant period of time did not expire between eachdelivery, such that the agent and expression construct would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other, with a delay time of only about12 hours being most preferred. In some situations, it may be desirableto extend the time period for treatment significantly, however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either theantibody with dual specificity for PD-L1 and PD-L2 or the other agentwill be desired. Various combinations may be employed, where a DiPDLantibody according to the present disclosure therapy is “A” and theother therapy is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Administration of the therapeutic agents of the present invention to apatient will follow general protocols for the administration of thatparticular secondary therapy, taking into account the toxicity, if any,of the antibody treatment. It is expected that the treatment cycleswould be repeated as necessary. It also is contemplated that variousstandard therapies, as well as surgical intervention, may be applied incombination with the described cancer therapies.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, Chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

1. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, Temazolomide (an aqueous form of DTIC), or any analog orderivative variant of the foregoing. The combination of chemotherapywith biological therapy is known as biochemotherapy. The presentinvention contemplates any chemotherapeutic agent that may be employedor known in the art for treating or preventing cancers.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as y-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic agent and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

3. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T-cells and NK cells. The combination of therapeuticmodalities, i.e., direct cytotoxic activity and inhibition or reductionof Fortilin would provide therapeutic benefit in the treatment ofcancer.

Immunotherapy could also be used as part of a combined therapy. Thegeneral approach for combined therapy is discussed below. In one aspectof immunotherapy, the tumor cell must bear some marker that is amenableto targeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155. An alternative aspect of immunotherapy is toanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growthfactors such as FLT3 ligand. Combining immune stimulating molecules,either as proteins or using gene delivery in combination with a tumorsuppressor such as mda-7 has been shown to enhance anti-tumor effects(Ju et al., 2000).

As discussed earlier, examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. Nos. 5,801,005; 5,739,169; Hui and Hashimoto, 1998;Christodoulides et al., 1998), cytokine therapy (e.g., interferons, and;IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson et al., 1998;Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, p53) (Qin etal., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945) and monoclonal antibodies (e.g., anti-ganglioside GM2,anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998;U.S. Pat. No. 5,824,311). Herceptin (trastuzumab) is a chimeric(mouse-human) monoclonal antibody that blocks the HER2-neu receptor. Itpossesses anti-tumor activity and has been approved for use in thetreatment of malignant tumors (Dillman, 1999). Combination therapy ofcancer with herceptin and chemotherapy has been shown to be moreeffective than the individual therapies. Thus, it is contemplated thatone or more anti-cancer therapies may be employed with thetumor-associated HLA-restricted peptide therapies described herein.

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders was few compared to those who did notrespond.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Human monoclonal antibodies are employed in passive immunotherapy, asthey produce few or no side effects in the patient. However, theirapplication is somewhat limited by their scarcity and have so far onlybeen administered intralesionally.

Human monoclonal antibodies to ganglioside antigens have beenadministered intralesionalty to patients suffering from cutaneousrecurrent melanoma (Irie & Morton, 1986). Regression was observed in sixout of ten patients, following, daily or weekly, intralesionalinjections. In another study, moderate success was achieved fromintralesional injections of two human monoclonal antibodies (Irie etal., 1989). Possible therapeutic antibodies include anti-TNF, anti-CD25,anti-CD3, anti-CD20, CTLA-4-IG, and anti-CD28.

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as described by Bajorin et al.(1988). The development of human monoclonal antibodies is described infurther detail elsewhere in the specification.

4. Gene Therapy

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the tumor-associated HLA-restricted peptide isadministered. Delivery of a vector encoding a the tumor-associatedHLA-restricted peptide in conjunction with a second vector encoding oneof the following gene products will have a combinedanti-hyperproliferative effect on target tissues. Alternatively, asingle vector encoding both genes may be used. A variety of proteins areencompassed within the invention, some of which are described below.Various genes that may be targeted for gene therapy of some form incombination with the present invention are well known to one of ordinaryskill in the art and may comprise any gene involved in cancers.

Inducers of Cellular Proliferation. The proteins that induce cellularproliferation further fall into ⁻various categories dependent onfunction. The commonality of all of these proteins is their ability toregulate cellular proliferation. For example, a form of PDGF, the sisoncogene, is a secreted growth factor. Oncogenes rarely arise from genesencoding growth factors, and at the present, sis is the only knownnaturally-occurring oncogenic growth factor. In one embodiment of thepresent invention, it is contemplated that anti-sense mRNA directed to aparticular inducer of cellular proliferation is used to preventexpression of the inducer of cellular proliferation.

The proteins FMS, ErbA, ErbB and neu are growth factor receptors.Mutations to these receptors result in loss of regulatable function. Forexample, a point mutation affecting the transmembrane domain of the Neureceptor protein results in the neu oncogene. The erbA oncogene isderived from the intracellular receptor for thyroid hormone. Themodified oncogenic ErbA receptor is believed to compete with theendogenous thyroid hormone receptor, causing uncontrolled growth.

The largest class of oncogenes includes the signal transducing proteins(e.g., Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity. Theproteins Jun, Fos and Myc are proteins that directly exert their effectson nuclear functions as transcription factors.

Inhibitors of Cellular Proliferation. The tumor suppressor oncogenesfunction to inhibit excessive cellular proliferation. The inactivationof these genes destroys their inhibitory activity, resulting inunregulated proliferation. The most common tumor suppressors are Rb,p53, p21 and p16. Other genes that may be employed according to thepresent invention include APC, DCC, NF-1, NF-2, WT-1, MEN4, MEN-II,zac1, p73, VHL, C-CAM, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16fusions, and p21/p27 fusions.

Regulators of Programmed Cell Death. Apoptosis, or programmed celldeath, is an essential process for normal embryonic development,maintaining homeostasis in adult tissues, and suppressing carcinogenesis(Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like proteaseshave been demonstrated to be important regulators and effectors ofapoptosis in other systems. The Bcl-2 protein, discovered in associationwith follicular lymphoma, plays a prominent role in controllingapoptosis and enhancing cell survival in response to diverse apoptoticstimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al.,1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Theevolutionarily conserved Bcl-2 protein now is recognized to be a memberof a family of related proteins, which can be categorized as deathagonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins thatshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

5. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, or 12 months. Thesetreatments may be of varying dosages as well.

V. ANTIBODY CONJUGATES

Antibodies may be linked to at least one agent to form an antibodyconjugate. In order to increase the efficacy of antibody molecules asdiagnostic or therapeutic agents, it is conventional to link orcovalently bind or complex at least one desired molecule or moiety. Sucha molecule or moiety may be, but is not limited to, at least oneeffector or reporter molecule. Effector molecules comprise moleculeshaving a desired activity, e.g., immunosuppression/anti-inflammation.Non-limiting examples of such molecules are set out above. Suchmolecules are optionally attached via cleavable linkers designed toallow the molecules to be released at or near the target site.

By contrast, a reporter molecule is defined as any moiety which may bedetected using an assay. Non-limiting examples of reporter moleculeswhich have been conjugated to antibodies include enzymes, radiolabels,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, photoaffinity molecules, colored particles orligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging.” Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imagingmoieties used can be paramagnetic ions, radioactive isotopes,fluorochromes, NMR-detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹ chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵ sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies may be produced according to well-knownmethods in the art. For instance, monoclonal antibodies can be iodinatedby contact with sodium and/or potassium iodide and a chemical oxidizingagent such as sodium hypochlorite, or an enzymatic oxidizing agent, suchas lactoperoxidase. Monoclonal antibodies may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups are often used to bind radioisotopes toantibody and exist as metallic ions are diethylenetriaminepentaaceticacid (DTPA) or ethylene di aminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodatnine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated are those intendedprimarily for use in vitro, where the antibody is linked to a secondarybinding ligand and/or to an enzyme (an enzyme tag) that will generate acolored product upon contact with a chromogenic substrate. Examples ofsuitable enzymes include urease, alkaline phosphatase, (horseradish)hydrogen peroxidase or glucose oxidase. Preferred secondary bindingligands are biotin and avidin and streptavidin compounds. The use ofsuch labels is well known to those of skill in the art and aredescribed, for example, in U.S. Pat. Nos. 3,817,837, 3,850,752,3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter and Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; Dholakia et al., 1989) and may be used as antibodybinding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaa.cetic acid anhydride(DTPA) ;ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may alsobe reacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors isachieved using monoclonal antibodies and the detectable imaging moietiesare bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulthydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

VI. IMMUNODETECTION METHODS

In still further embodiments, there are immunodetection methods forbinding, purifying, removing, quantifying and otherwise generallydetecting PD-L1 and PD-L2 and their associated antigens. Someimmunodetection methods include enzyme lurked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot to mention a few. In particular, a competitive assay forthe detection and quantitation of antibodies with dual specificity toPD-L1 and PD-L2 also is provided. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis andGaland (1993), De Jager et al. (1993), and Nakamura et al. (1987). Ingeneral, the immunobinding methods include obtaining a sample andcontacting the sample with a first antibody in accordance withembodiments discussed herein, as the case may be, under conditionseffective to allow the formation of immunocomplexes.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to PD-L1 andPD-L2 present in the sample. After this time, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or Westernblot, will generally be washed to remove any non-specifically boundantibody species, allowing only those antibodies specifically boundwithin the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. Patents concerning the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149 and 4,366,241. Of course, one may find additionaladvantages through the use of a secondary binding ligand such as asecond antibody and/or a biotin/avidin ligand binding arrangement, as isknown in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding ligand, such as an antibody that hasbinding affinity for the antibody, is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firstbiotinylated antibody is used to detect the target antigen, and a secondantibody is then used to detect the biotin attached to the complexedbiotin. In that method, the sample to be tested is first incubated in asolution containing the first step antibody. If the target antigen ispresent, some of the antibody binds to the antigen to form abiotinylated antibody/antigen complex. The antibody/antigen complex isthen amplified by incubation in successive solutions of streptavidin (oravidin), biotinylated. DNA, and/or complementary biotinylated DNA, witheach step adding additional biotin sites to the antibody/a.ntigencomplex. The amplification steps are repeated until a suitable level ofamplification is achieved, at which point the sample is incubated in asolution containing the second step antibody against biotin. This secondstep antibody is labeled, as for example with an enzyme that can be usedto detect the presence of the antibody/antigen complex byhistoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

A. ELISAs

Immunoassays, in their most simple sense, are binding assays. Certainpreferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining PD-L1 and/or PD-L2 is added to the wells. After binding andwashing to remove non-specifically bound immune complexes, the boundantigen may be detected. Detection may be achieved by the addition ofanother dual specificity PD-L1 and PD-L2 antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA.”Detection may also be achieved by the addition of a second dualspecificity PD-L1 and PD-L2 antibody, followed by the addition of athird antibody that has binding affinity for the second antibody, withthe third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing thePD-L1 or PD-L2 antigens are immobilized onto the well surface and thencontacted with dual specificity antibodies for PD-L1 and PD-L2 (DiPDL).After binding and washing to remove non-specifically bound immunecomplexes, the bound dual specificity antibodies are detected. Where theinitial DiPDL antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstantibody, with the second antibody being linked to a detectable label,

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder.

The coating allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween)

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ARTS),or H202, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analyticaltechnique used to detect specific proteins in a given sample of tissuehomogenate or extract. It uses gel electrophoresis to separate native ordenatured proteins by the length of the polypeptide (denaturingconditions) or by the 3-D structure of the protein(native/non-denaturing conditions). The proteins are then transferred toa membrane (typically nitrocellulose or PVDF), where they are probed(detected) using antibodies specific to the target protein.

Samples may be taken from whole tissue or from cell culture. In mostcases, solid tissues are first broken down mechanically using a blender(for larger sample volumes), using a homogenizer (smaller volumes), orby sonication. Cells may also be broken open by one of the abovemechanical methods. However, it should be noted that bacteria, virus orenvironmental samples can be the source of protein and thus Westernblotting is not restricted to cellular studies only. Assorteddetergents, salts, and buffers may be employed to encourage lysis ofcells and to solubilize proteins. Protease and phosphatase inhibitorsare often added to prevent the digestion of the sample by its ownenzymes. Tissue preparation is often done at cold temperatures to avoidprotein denaturing.

The proteins of the sample are separated using gel electrophoresis.Separation of proteins may be by isoelectric point (pI), molecularweight, electric charge, or a combination of these factors. The natureof the separation depends on the treatment of the sample and the natureof the gel. This is a very useful way to determine a protein. It is alsopossible to use a two-dimensional (2-D) gel which spreads the proteinsfrom a single sample out in two dimensions. Proteins are separatedaccording to isoelectric point (pH at which they have neutral netcharge) in the first dimension, and according to their molecular weightin the second dimension.

In order to make the proteins accessible to antibody detection, they aremoved from within the gel onto a membrane made of nitrocellulose orpolyvinylidene difluoride (PVDF). The membrane is placed on top of thegel, and a stack of filter papers placed on top of that. The entirestack is placed in a buffer solution which moves up the paper bycapillary action, bringing the proteins with it. Another method fortransferring the proteins is called electroblotting and uses an electriccurrent to pull proteins from the gel into the PVDF or nitrocellulosemembrane. The proteins move from within the gel onto the membrane whilemaintaining the organization they had within the gel. As a result ofthis blotting process, the proteins are exposed on a thin surface layerfor detection (see below). Both varieties of membrane are chosen fortheir non-specific protein binding properties (i.e., binds all proteinsequally well). Protein binding is based upon hydrophobic interactions,as well as charged interactions between the membrane and protein.Nitrocellulose membranes are cheaper than PVDF, but are far more fragileand do not stand up well to repeated probing. The uniformity and overalleffectiveness of transfer of protein from the gel to the membrane can bechecked by staining the membrane with Coomassie Brilliant Blue orPonceau S dyes. Once transferred, proteins are detected using labeledprimary antibodies, or unlabeled primary antibodies followed by indirectdetection using labeled protein A or secondary labeled antibodiesbinding to the Fc region of the primary antibodies.

C. Immunohistochemistry

The antibodies may also be used in conjunction with both fresh-frozenand/or formalin-fixed, paraffm-embedded tissue blocks prepared for studyby immunohistochemistry (IHC). The method of preparing tissue blocksfrom these particulate specimens has been successfully used in previousIHC studies of various prognostic factors, and is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred elal., 1990),

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections fromthe capsule. Alternatively, whole frozen tissue samples may be used forserial section cuttings.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections. Again, whole tissue samples may besubstituted.

D. Immummodetection Kits

In still further embodiments, there are immunodetection kits for usewith the immunodetection methods described above. The immunodetectionkits will thus comprise, in suitable container means, a first antibodythat binds to PD-L1 or PD-L2 antigens, and optionally an immunodetectionreagent.

In certain embodiments, the dual specificity PD-L1 and PD-L2 antibodymay be pre-bound to a solid support, such as a column matrix and/or wellof a microtiter plate. The immunodetection reagents of the kit may takeany one of a variety of forms, including those detectable labels thatare associated with or linked to the given antibody. Detectable labelsthat are associated with or attached to a secondary binding ligand arealso contemplated. Exemplary secondary ligands are those secondaryantibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and all such labels may beemployed in connection with embodiments discussed herein.

The kits may further comprise a suitably aliquoted composition of thePD-L1 and PD-L2 antigens, whether labeled or unlabeled, as may be usedto prepare a standard curve for a detection assay. The kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, or as separate moieties to be conjugated by the userof the kit. The components of the kits may be packaged either in aqueousmedia or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody may be placed, or preferably, suitably aliquoted. Thekits will also include a means for containing the antibody, antigen, andany other reagent containers in close confinement for commercial sale.Such containers may include injection or blow-molded plastic containersinto which the desired vials are retained.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention, it should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Materials and Methods

Antibody selection, generation, and production. Although additionaldetail may be provided in subsequent Examples, the selection,generation, and production of the disclosed antibodies were performedgenerally as follows.

Antigen production—Antigens were biotinylated using the EZ-LinkSulfo-NHS-Biotinylation Kit from Pierce. Goat F(ab′)₂ anti-humankappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE) and Streptavidin-AF633(SA-633) were obtained from Southern Biotech, Sigma, and MolecularProbes, respectively. Streptavidin MicroBeads and MACS LC separationcolumns were purchased from Miltenyi Biotec. Goat anti-human IgG-PE(Human-PE) was obtained from Southern Biotech.

In order to generate the desired cross-reactive antibodies, selectionsas described in more detail below, using each target antigen,independently, were alternated from round-to-round to enrich for thedual specificity. More specifically, PD-L1 and PD-L2 antibody heavychains from four rounds of naïve selection were paired with 9 distinctcommon light chain libraries (cLC) and selected for PD-L1 and PD-L2binding antibodies (FIG. 1). These antibodies were that subjected toaffinity maturation using CDR-H1 and CDR-H2 libraries to yield the finalclones (FIG. 1).

Naïve Discovery—Eight naïve human synthetic yeast libraries each of ˜10⁹diversity were propagated as previously described (see, i, Xu et al.,2013; WO2009036379; WO2010105256; and WO2012009568.) For the first tworounds of selection, a magnetic bead sorting technique utilizing theMiltenyi MACS system was performed, as previously described (see, e.g.,Siegel et al, 2004). Briefly, yeast cells (˜10¹⁰ cells/library) wereincubated with 3 ml of 10 biotinylated Fc fusion-antigen for 15 min at30° C. in wash buffer (phosphate-buffered saline (PBS)/0.1% bovine serumalbumin (BSA)). After washing once with 40 ml ice-cold wash buffer, thecell pellet was resuspended in 20 mL wash buffer, and StreptavidinMicroBeads (500 were added to the yeast and incubated for 15 min at 4°C. Next, the yeast were pelleted, resuspended in 20 mL wash buffer, andloaded onto a Miltenyi LS column. After the 20 mL were loaded, thecolumn was washed 3 times with 3 ml wash buffer. The column was thenremoved from the magnetic field, and the yeast were eluted with 5 mL ofgrowth media and then grown overnight. The following rounds of selectionwere performed using flow cytometry. Approximately 2×10⁷ yeast werepelleted, washed three times with wash buffer, and incubated at 30° C.with either 10 nM Fc-fusion antigen or in later rounds decreasingconcentrations of biotinylated antigen (100 to 1 nM) under equilibriumconditions, 100 nM biotinylated antigens of different species (mouse) inorder to obtain species cross-reactivity, or with a poly-specificitydepletion reagent (PSR) to remove non-specific antibodies from theselection. For the PSR depletion, the libraries were incubated with a1:10 dilution of biotinylated PSR reagent as previously described (see,e.g., Xu et al., 2013). Yeast were then washed twice with wash bufferand stained with LC-FITC (diluted 1:100) and either SA-633 (diluted1:500) or EAPE (diluted 1:50) secondary reagents for 15 min at 4° C.After washing twice with wash buffer, the cell pellets were resuspendedin 0.3 mL wash buffer and transferred to strainer-capped sort tubes.Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sortgates were determined to select for antibodies with desiredcharacteristics. Selection rounds were repeated until a population withall of the desired characteristics was obtained.

In order to generate cross-reactive antibodies, target antigens werealternated from round-to-round to enrich for the dual specificity. Afterthe final round of sorting, yeast were plated and individual colonieswere picked for characterization.

Light chain hatch shuffle (LCBS)—The primary discovery also included alight chain batch diversification protocol from heavy chain plasmidsfrom the naïve selections:

Heavy chain plasmids from a naïve round four selection output wereextracted from the yeast and transformed into a light chain library witha diversity of 5×10⁶. Selections were performed with one round of MACSand three rounds of FACS employing the same conditions as the naïvediscovery.

Antibody Optimization—Optimization of antibodies was performed byintroducing diversities into the heavy chain and light chain variableregions as described below. A combination of some of these approacheswas used for each antibody.

CDRH1 and CDRH2 selection: The CDRH3 of a single antibody was recombinedinto a premade library with CDRH1 and CDRH2 variants of a diversity of1×10⁸ and selections were performed with one round of MACS and fourrounds of FACS as described in the naïve discovery. In the FACS roundsthe libraries were looked at for PSR binding, species cross-reactivity,antigen cross-reactivity and affinity pressure, and sorting wasperformed in order to obtain a population with the desiredcharacteristics. For these selections affinity pressures were appliedeither by titrating down biotinylated monomeric antigen or bypreincubating the biotinylated antigen with parental Fab for 30 minutesand then applying that precomplexed mixture to the yeast library for alength of time which would allow the selection to reach an equilibrium.The higher affinity antibodies were then able to be sorted.

VH Mut selection: The heavy chain variable region (VH) was mutagenizedvia error prone PGR. The library was then created by transforming thismutagenized VH and the heavy chain expression vector into yeast alreadycontaining the light chain plasmid of the parent. Selections wereperformed similar to previous cycles using FACS sorting for threerounds. In the FACS rounds the libraries were looked at forcross-reactivity and affinity pressure, and sorting was performed inorder to obtain a population with the desired characteristics.

CDRL1, CDRL2 and CDRL3 selection: Oligos were ordered from IDT whichcomprised the CDRL3 and were variegated via NNK diversity. The CDRL3oligos were double-stranded using primers which annealed to the flankingregion of the CDRL3. These double-stranded CDRL3 oligos were thenrecombined into a premade library with CDRL1 and CDRL2 variants of adiversity of 3×10⁵ and selections were performed with one round of MACSand three rounds of FACS as described in the naïve discovery. In theFACS rounds the libraries were looked at for PSR binding,cross-reactivity, and affinity pressure, and sorting was performed inorder to obtain a population with the desired characteristics. Affinitypressures for these selections were performed as described above in theCDRH1 and CDRH2 selection and alternating antigens from round to roundwas applied to enrich for dual binders.

Antibody production and purification—Yeast clones were grown tosaturation and then induced for 48 h at 30° C. with shaking. Afterinduction, yeast cells were pelleted and the supernatants were harvestedfor purification. IgGs were purified using a Protein A column and elutedwith acetic acid, pH 2.0. Fab fragments were generated by papaindigestion and purified over KappaSelect (GE Healthcare LifeSciences)

ForteBio K_(D) measurements—ForteBio affinity measurements wereperformed on an Octet RED384 generally as previously described (see,e.g., Estep et al., 2013). Briefly, ForteBio affinity measurements wereperformed by loading IgGs on-line onto AHQ sensors. Sensors wereequilibrated off-line in assay buffer for 30 min and then monitoredon-line for 60 seconds for baseline establishment. Sensors with loadedIgGs were exposed to 100 nM antigen for 3 minutes, and afterwards weretransferred to assay buffer for 3 min for off-rate measurement. Formonovalent affinity assessment Fabs were used instead of IgGs. For thisassessment the unbiotinylated Fc fusion antigen was loaded on-line ontothe AHQ sensors. Sensors were equilibrated off-line in assay buffer for30 min and then monitored on-line for 60 seconds for baselineestablishment. Sensors with loaded antigen were exposed to 100 nM Fabfor 3 minutes, and afterwards they were transferred to assay buffer for3 min for off-rate measurement. All kinetics were analyzed using the 1:1binding model.

ForteBio Epitope Binning/Ligand Blocking—Epitope binning/ligand blockingwas performed using a standard sandwich format cross-blocking assay.Control anti-target IgG was loaded onto AHQ sensors and unoccupiedFc-binding sites on the sensor were blocked with an irrelevant humanIgG1 antibody. The sensors were then exposed to 100 nM target antigenfollowed by a second anti-target antibody or ligand. Additional bindingby the second antibody or ligand after antigen association indicates anunoccupied epitope (non-competitor), while no binding indicates epitopeblocking (competitor or ligand blocking).

MSD-SET K_(D) measurements—Equilibrium affinity measurements of selectedhigh affinity antibodies were performed generally as previouslydescribed (Estep et al., 2013). Briefly, solution equilibrium titrations(SET) were performed in PBS +0.1% IgG-Free BSA (PBSF) with antigen heldconstant at 50 pM and incubated with 3- to 5-fold serial dilutions ofFab starting at 20 nM. Antibodies (2.0 nM in PBS) were coated ontostandard bind MSD-ECL plates overnight at 4° C. or at room temperaturefor 30 min. Plates were then blocked by BSA for 30 min with shaking at700 rpm, followed by three washes with wash buffer (PBSF+0.05% Tween20). SET samples were applied and incubated on the plates for 150s withshaking at 700 rpm followed by one wash. Antigen captured on a plate wasdetected with 250 ng/mL sulfotag-labeled streptavidin in PBSF byincubation on the plate for 3 min. The plates were washed three timeswith wash buffer and then read on the MSD Sector Imager 2400 instrumentusing 1× Read Buffer T with surfactant. The percent free antigen wasplotted as a function of titrated antibody in Prism and fit to aquadratic equation to extract the K_(D). To improve throughput, liquidhandling robots were used throughout MSD-SET experiments, including SETsample preparation.

Cell Binding Analysis—Approximately 100,000 cells overexpressing theantigen were washed with wash buffer and incubated with 100 μl 100 nMIgG for 5 minutes at room temperature. Cells were then washed twice withwash buffer and incubated with 100 μl of 1:100 Human-PE for 15 minuteson ice. Cells were then washed twice with wash buffer and analyzed on aFACS Canto II analyzer (BD Biosciences)

Antibody Characterization. Antibody candidates generated from thepresentation methods described above were tested for the capacity tobind to PD-L1 or PD-L2. To generate affinity K_(D) to human PD-L1 andPD-L2, candidate monoclonal Abs were loaded onto Anti-Human Fc Capture(AHC) Biosensors at 100 nM (15 ug/mL), and human PD-L1 or PD-L2 proteinassociation and dissociation was tested in dilution series from 30-0.37nM. The binding and release of the analyte (PD-L1 or PD-L2) are recordedby the Octet instrument in real time and then used to calculate theK_(D), K_(on), and K_(dis); results are derived from 2:1 Global FitModeling with reference well subtraction. To generate affinity K_(D) tomurine PD-L1 and PD-L2, candidate antibodies were covalently immobilizedonto activated Amine Reactive 2nd Generation (AR2G) Biosensors (quenchedwith 1M ethanolamine 8.5 after protein loading) at 100 nM (15 p.g/mL),and mouse PD-L1 or PD-L2 protein association and dissociation was testedin dilution series from 300-1 nM. The binding and release of the analyteare recorded by the Octet instrument in real time and then used tocalculate the K_(D), K_(on), and K_(dis); results are derived from 2:1Global Fit Modeling with reference well subtraction.

Candidate antibodies prevent PD-1:PD-L1, PD-1:PD-L2, andPD-1:PD-L1/PD-L2 binding. Candidate PD-L1, PD-L2, or DiPDL monoclonalantibodies and FDA approved antibodies were assayed using the Promega®PD-L1, PD-L2, or PD-L1/PD-PD-1 blockade systems. Varying concentrationsof antibody were added to CHO-PD-L1, CHO-PD-L2, or CHO-PD-L1/PD-L2cells. PD-1 effector cells are Jurkat T cells which can be stimulated bythe CHO-PD-L1, CHO-PD-L2, or CHC_)-PD-L1/PD-L2 cells. The PD-1 effectorcells, which produce firefly Luciferase in response to activation, wereincubated with antibody and CHO cells for 6 hours and then the resultswere read out on a luminometer using the Bio-glo™ assay kit (Promega®)according to the manufacturer's instructions. Blocking was assessed asan increase in luciferase signal. Analysis was conducted using GraphPadPrism® software.

DiPDL antibody activity in mixed lymphocyte reactions. CD14+ monocyteswere isolated from peripheral blood mononuclear cells using CD14microbeads. Cells were seeded at 1 million/ml in and stimulated withIL-4 and GM-CSF in 10% FCS/RPMI/P/S cell culture medium. Cells werecultured for 7 days to differentiate into immature dendritic cells(1DCs) and varying concentrations of PD-L1, both PD-L1 and PD-L2, DiPDLor commercial antibodies were added. IDCs were then used to stimulateCD4+ cells at a ratio of 10:1 CD4:IDCs, IFN-γ was assayed by ELISAfollowing the protocols provided by R&D systems.

Antibody Activity against Human PD-L1/L2+ Lymphoma. Varyingconcentrations of candidate PD-L1, PD-L2, or DiPDL antibodies (humanIgG1) were added with in vitro expanded human PBMC to Calcein(Thermaisher®)-labelled U2940 PMBL cells at an effector to target ratioof 15 to 1. Percent specific lysis was calculated as the differencebetween experimental release and spontaneous release of Calcein asmeasured on a fluorescent plate reader.

Antibody activity against xenograft tumors. U2940 PMBL xenograft tumorswere established in immunodeficient mice. Tumors were allowed to reach avolume of 150 mm³. After reaching 150 mm³, the mice were treated withthe antibody therapeutics shown twice per week, at a treatment of 10mg/kg for 3 weeks with 9 mice per treatment group. Caliper measurementsof tumor width, length, and depth were used to calculate tumor volume.

Example 2—Results

Selection of Multiple Families of PD-L1/PD-L2 Antibodies Sharing aCommon Light Chain. PD-L1 and PD-L2 heavy chains from several rounds ofselections as described in Example 1 were paired with 9 distinct commonlight chain libraries (cLC) and selected for PD-L1 and PD-L2 binding.Those which showed PD-L1 and PD-L2 binding were then subjected toaffinity maturation. Clones ADI-20225 and ADI-20236 can be paired toform a bispecific antibody sharing a Vk1-l2 light chain (FIG. 1). ClonesADI-20230 and AM-20241 can be paired to form a PD-L1/PD-L2 bispecificantibody sharing a Vk11-05 light chain (FIG. 1).

Equivalence of DiPDL Antibodies to FDA-approved Antibodies for BlockingPD-L1 or PD-L2 Binding to PD-1. Candidate antibody clones cLC PD-L1, cLCPD-L2, or assembled DiPDL monoclonal antibodies identified as describedin Example 1 above were assayed using the Promega® PD-L1 and PD-L2blocking assay, for their ability to block the binding of PD-L1 andPD-L2 to PD-1. It was found that, compared to an isotype control,candidate PD-L1 antibodies 20225 and 20230 effectively sequestered PD-L1from PD-1, thereby inducing the production of firefly luciferase (Ha 2).Similarly, it was found that, compared to an isotype control, candidatePD-L2 antibodies 20236 and 20241 effectively sequestered PD-L2 fromPD-1, thereby inducing the production of firefly luciferase (FIG. 2).DiPDL candidates C1 (having the 20225 light chain and 20236 heavychain), and C2 (having the 20230 heavy chain and 20241 light chain)effectively prevented PD-L1 and PD-L2 binding to PD-1 as well asKeytnida (an anti-PD-1 antibody). The candidate antibodies inducedluciferase production at least equivalently to approvedimmunotherapeutics: Keytruda, Tecentriq, and Avelumab (FIG. 2).

Candidate Antibodies Are Active Across Multiple Human Mixed LymphocyteReactions. Candidate antibodies were evaluated in the presence ofinduced dendritic cells and T-cells from separate donors. CandidateDiPDL antibody 22726 was found to induce IFN-γ production at least aswell as FDA approved Keytruda and Tecentriq, and as well as PD-L1candidate 20230 alone or 20230 and candidate PI)-L2 antibody 20241combined (FIG. 3).

Candidate Antibodies Mediate Efficient ADCC Against Human PD-L1/L2+Lymphoma. Varying concentrations of candidate PD-L1, PD-L2, or DiPDL,antibodies (human IgG1), or Rituxan were added with in vitro expandedhuman PBMCs to Calcein-labelled U2940 PMBL cells at an effector totarget ratio of 15 to 1 to stimulate ADCC. The IgG1 control showed noincrease in percent lysis. Whereas FDA approved antibody Rituxanincreased the percentage of cells lysed to about 50% (FIG. 4). Theaddition of the candidate antibodies triggered cell lysis of betweenabout 50% up to greater than 75% of cells, with the DiPDL antibodytriggering the greatest percentage of lysis of those antibodies tested(FIG. 4).

Candidate Antibodies With ADCC Are Active Against Human U2940 Lymphomain vivo. Following establishment of PBML xenograft tumors in SCID mice,mice were treated with either Herceptin, Rituxan, Avelumab and DiPDLcandidate 22726. Each was evaluated for its ability to inhibit tumorgrowth. The DiPDL candidate was shown to have stronger inhibitoryactivity on tumor growth than either Herceptin or Avelumab (FIG. 5).

Candidate Antibodies Avidly Bind Human PD-L1, and PD-L2. Avidity curveswere generated for subclones 20225, 20230, 20236, and 20241 on theForteBio Octet®. Antibody subclones were immobilized on Anti-Human FcCapture (AHC) Biosensors at 100 nM (15 μg/mL), and human PD-L1 and PD-L2protein association and dissociation was tested in dilution series from30-0.37 nM. Affinity was calculated for each of the subclones from thedata and is presented in Table 4. The PD-L1 antibody subclones avidlybind human PD-L1, while the PD-L2 antibody subclones avidly bind PD-L2.

TABLE 1 NUCLEIC ACID SEQUENCES FOR ANTIBODY VARIABLE REGIONS SEQ IDClone Variable Sequence Region NO: 20225CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT 3 HeavyCAGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCCGTAGCTATGCTATCCAGTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCATCCCTGGTTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTAGATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGAGAGTTGTCCCACGGTGGTCTCAGTTACTGGGGACAGGGTACATTGGTCA CCGTCTCCTCA 20225GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG 4 LightACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCTATCACCTTTGGCGGAGGGACCAAGGTTGAGATCAAA 20230CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT 5 HeavyCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATTTTATCTCGTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGACAGATCATCCCTAATGTGGGTAGAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGGGACCAGACCGCTAAGGCACACCTGGTCTGGGGACAGGGTACATTGGTCA CCGTCTCCTCA 20230GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAG 6 LightACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTAGTGCCAACAGTATAATAGTTATCCTATCACCTTGGCGGAGGGACCAAGGTTGAGATCAAA 20236CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT 7 HeavyCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCTCGAGCCATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTTGGGCATTGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTAGTAGTGCGCCAGACCAGGAGACCACAGAGCTTCCCATTTCGATTACTGGGGACAGGGTACAT TGGTCACCGTCTCCTCA20236 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG 8 LightACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCTATCACCTTTGGCGGAGGGACCAAGGTTGAGATCAAA 20241CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCT 9 HeavyCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCGAGTATGCTATCGTGTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTCTGTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGACCGTTGGACCGCAGAATAGTAGCATTCGACATATGGGGTCAGGGTACAA TGGTCACCGTCTCCTCA20241 GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAG 10 LightACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATCCTATCACCTTTGGCGGAGGGACCAAGGTTGAGATCAAA

TABLE 2 AMINO ACID SEQUENCES FOR ANTIBODY VARIABLE REGIONS SEQ ID CloneVariable Sequence NO: 20225QVQLVQSGAEVKKPGASVKVSCKASGGTFRSYAIQKVRQAPGQGLEWMG 11 HeavyWIIPGFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR ELSHGGLSYWGQGTLVTVSS20225 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIY 12 LightAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITF GGGTKVEIK 20230QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYFISWVRQAPGQGLEWMG 13 HeavyQIIPNVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR DQTAKAHLVWGQGTLVTVSS20230 DIQMTQSPSTLSASVGDRVTITCRASQSISSKLAWYQQKPGKAPKLLIY 14 LightKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPITF GGGTKVEIK 20236QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHAISWVRQAPGQGLEWMG 15 HeavyGIIPIFGWALYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR PGDKRASKFDYWGQGTLVTVSS20236 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIY 16 LightAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITF GGGTKVEIK 20241QVQLVQSGAEVKKPGSSVKVSCKASGGTFSEYAIVWVRQAPGQGLEWMG 17 HeavyGIIPLFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR PLDRRIVAFDIWGQGTMVTVSS20241 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY 18 LightKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPITF GGGTKVEIK

TABLE 3 CDR SEQUENCES CDR1 CDR2 CDR3 Anti- (SEQ (SEQ (SEQ body ChainID NO:) ID NO:) ID NO:) 20225 Heavy GTFRSYAIQ WIIPGFGTANY ARELSHGGL (19)AQKFQG (20) SY (21) 20225 Light RASQGISSW AASSLQS QQANSFPIT LA( 22) (23)(24) 20230 Heavy GTFSSYFIS QIIPNVGTANY ARDQTAKAH (25) AQKFQG (26)LV (27) 20230 Light RASQSISSW KASSLES QQYNSYPIT LA (23) (29) (30) 20236Heavy GTFSSHAIS GI1PIFGWALY ARFGDHRAS (31) AQKFQG (32) HFDY (33) 20236Light RASQGISSW AASSLQS QQANSFPIT LA (34) (35) (36) 20241 HeavyGTFSEYAIV GIIPLFGTANY ARPLDRRIV (37) AQKFQG (38) AFDI (39) 20241 LightRASQSISSW KASSLES QQYNSYPIT LA (40) (41) (42)

TABLE 4 AFFINITY OF ANTIBODY BINDING TO PD-L1 AND PD-L2 ForteBioForteBio ForteBio Species IgG KD Human IgG KD Cyno IgG KD Mouse LineageORBIT ADI Binding PD1 CD80 cross- PD-L1-Fc PD-L1-Fc PD-L1-Fc (D

PDL) Name Name profile competitor competitor reactivity (M) Avid (M)Avid (M) Avid cLC-PDL1 (22731) PD-L1-C1-1 ADI-20225

Yes Yes Cyno-Mouse 2.23E−10 2.27E−10 P.F. cLC-PDL1 (22726) PD-L1-C2-1ADI-20230

Yes Yes Cyno 2.05E−10 2.84E−10

cLC-PDL2 (22731) PD-L2-C1-1 ADI-20236

Yes N.A Cyno-Mouse

cLC-PDL2 (22726) PD-L2-C2-1 ADI-20241

Yes N.A Cyno-mouse

ForteBio ForteBio ForteBio ForteBio ForteBio IgG KD Human IgG KD CynoIgG KD Mouse Fab KD Human Fab KD Human Lineage PD-L2-Fc PD-L2-FcPD-L2-Fc PD-L1-Fc PD-L2-Fc (D

PDL) (M) Avid (M) Avid (M) Avid (M) Monovalent (M) Monovalent cLC-PDL1(22731)

3.33E−10

cLC-PDL1 (22726)

8.23E−10

cLC-PDL2 (22731) 4.16E−10 6.40E−10 P.F.

5.41E−10 cLC-PDL2 (22726) 4.87E−10 2.53E−10 P.F.

1.99E−09

indicates data missing or illegible when filed

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VIII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An antibody or antibody fragment comprising a first set of clonepaired heavy and light CDR sequences selected from SEQ ID NOS: 19-24 or25-30, and a second set of clone paired heavy and light CDR sequencesselected from SEQ ID NOS: 31-36 or 37-42.
 2. The antibody or antibodyfragment of claim 1, wherein said antibody or antibody fragment isencoded by a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired of heavy and light chain variable sequences selected fromSEQ ID NOS: 7-8 or 9-10.
 3. The antibody or antibody fragment of claim1, wherein said antibody or antibody fragment is encoded by heavy andlight chain variable sequences having at least 70%, 80%, or 90% identityto a first set of clone paired of heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired of heavy and light chain variable sequences selected fromSEQ ID NOS: 7-8 or 9-10.
 4. The antibody or antibody fragment of claim1, wherein said antibody or antibody fragment is encoded by heavy andlight chain variable sequences having at least 95% identity to a firstset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 3-4 or 5-6, and a second set of clone paired heavy andlight chain variable sequences selected from SEQ ID NOS: 7-8 or 9-10. 5.The antibody or antibody fragment of claim 1, wherein said antibody orantibody fragment comprises a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18.
 6. The antibody or antibodyfragment of claim 1, wherein said antibody or antibody fragmentcomprises heavy and light chain variable sequences having at least 70%,80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18.
 7. The antibody or antibodyfragment of claim 1, wherein said antibody or antibody fragmentcomprises heavy and light chain variable sequences having 95% identityto a first set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18.
 8. The antibody or antibody fragment of claim 1,wherein the antibody fragment is a recombinant di-scFv (divalent singlechain fragment variable) antibody or F(ab′)₂ fragment.
 9. The antibodyor antibody fragment of claim 1, wherein said antibody is a chimericantibody.
 10. The antibody or antibody fragment of claim 1, wherein saidantibody is an IgG.
 11. The antibody or antibody fragment of claim 1,wherein said antibody or antibody fragment further comprises a cellpenetrating peptide and/or is an intrabody.
 12. The antibody or fragmentof claim 1, wherein said antibody or antibody fragment is a humanantibody.
 13. The antibody or fragment of claim 1, wherein said antibodyor antibody fragment is a humanized antibody.
 14. A method of treating asubject having cancer comprising delivering to said subject an antibodyor antibody fragment comprising a first set of clone paired heavy andlight CDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and asecond set of clone paired heavy and light CDR sequences selected fromSEQ ID NOS: 31-36 or 37-42.
 15. The method of claim 14, wherein theantibody or antibody fragment is encoded by a first set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 3-4or 5-6, and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10.
 16. The method of claim14, the antibody or antibody fragment is encoded by heavy and lightchain variable sequences having at least 70%, 80%, or 90% identity to afirst set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 3-4 or 5-6, and a second set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 7-8or 9-10.
 17. The method of claim 14, wherein said antibody or antibodyfragment is encoded by heavy and light chain variable sequences havingat least 95% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10.
 18. The method of claim 14,wherein said antibody or antibody fragment comprises a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18. 19.The method of claim 14, wherein said antibody or antibody fragmentcomprises heavy and light chain variable sequences having at least 70%,80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18.
 20. The method of claim 14,wherein said antibody or antibody fragment comprises heavy and lightchain variable sequences having at least 95% identity to a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18. 21.The method of claim 14, wherein the antibody fragment is a recombinantdi-scFv (divalent single chain fragment variable) antibody or F(ab′)₂fragment.
 22. The method of claim 14, wherein said antibody is an IgG.23. The method of claim 14, wherein said antibody is a chimericantibody.
 24. The method of claim 14, wherein delivering comprisesantibody or antibody fragment administration, or genetic delivery withan RNA or DNA sequence or vector encoding the antibody or antibodyfragment.
 25. A hybridoma or engineered cell encoding an antibody orantibody fragment wherein the antibody or antibody fragment ischaracterized by a first set of clone paired heavy and light CDRsequences selected from SEQ ID NOS: 19-24 or 25-30, and a second set ofclone paired heavy and light CDR sequences selected from SEQ ID NOS:31-36 or 37-42.
 26. The hybridoma or engineered cell of claim 25,wherein said antibody or antibody fragment is encoded by a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 3-4 or 5-6, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 7-8 or 9-10.
 27. Thehybridoma or engineered cell of claim 25, wherein said antibody orantibody fragment is encoded by heavy and light chain variable sequenceshaving at least 70%, 80%, or 90% identity to a first set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 3-4or 5-6, and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10.
 28. The hybridoma orengineered cell of claim 27, wherein said antibody or antibody fragmentis encoded by heavy and light chain variable sequences having at least95% identity to a first set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 3-4 or 5-6, and a secondset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 7-8 or 9-10.
 29. The hybridoma or engineered cell ofclaim 25, wherein said antibody or antibody fragment comprises a firstset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 11-12 or 13-14, and a second set of clone paired heavyand light chain variable sequences selected from SEQ ID NOS: 15-16 or17-18.
 30. The hybridoma or engineered cell of claim 25, wherein saidantibody or antibody fragment comprises heavy and light chain variablesequences having at least 70%, 80%, or 90% identity to a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 11-12 or 13-14, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 15-16 or 17-18. 31.The hybridoma or engineered cell of claim 25, wherein said antibody orantibody fragment comprises heavy and light chain variable sequenceshaving 95% identity to a first set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 11-12 or 13-14, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 15-16 or 17-18.
 32. The hybridoma orengineered cell of claim 25, wherein the antibody fragment is arecombinant di-scFv (divalent single chain fragment variable) antibodyor F(ab′)₂ fragment.
 33. The hybridoma or engineered cell of claim 25,wherein said antibody is a chimeric antibody.
 34. The hybridoma orengineered cell of claim 25, wherein said antibody is an IgG.
 35. Thehybridoma or engineered cell of claim 25, wherein said antibody orantibody fragment further comprises a cell penetrating peptide and/or isan intrabody.
 36. A vaccine formulation comprising antibodies orantibody fragments characterized by a first set of clone paired heavyand light CDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and asecond set of clone paired heavy and light CDR sequences selected fromSEQ ID NOS: 31-36 or 37-42.
 37. The vaccine formulation of claim 36,wherein said antibody or antibody fragment is encoded by a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 3-4 or 5-6, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 7-8 or 9-10.
 38. Thevaccine formulation of claim 36, wherein said antibody or antibodyfragment is encoded by heavy and light chain variable sequences havingat least 70%, 80%, or 90% identity to a first set of clone paired heavyand light chain variable sequences selected from SEQ ID NOS: 3-4 or 5-6,and a second set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 7-8 or 9-10.
 39. The vaccineformulation of claim 36, wherein said antibody or antibody fragment isencoded by heavy and light chain variable sequences having at least 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 3-4 or 5-6, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 7-8 or 9-10.
 40. The vaccine formulation of claim 36, whereinsaid antibody or antibody fragment comprises a first set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 11-12or 13-14, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 15-16 or 17-18.
 41. Thevaccine formulation of claim 36, wherein said antibody or antibodyfragment comprises heavy and light chain variable sequences having 95%identity to a first set of clone paired heavy and light chain variablesequences selected from SEQ ID NOS: 11-12 or 13-14, and a second set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 15-16 or 17-18.
 42. The vaccine formulation of claim 36, whereinat least one antibody fragment is a recombinant di-scFv (divalent singlechain fragment variable) antibody, Fab fragment, F(ab′)₂ fragment, or Fvfragment.
 43. The vaccine formulation of claim 36, wherein at least oneantibody is a chimeric antibody.
 44. The vaccine formulation of claim36, wherein at least one antibody is an IgG.
 45. The vaccine formulationof claim 36, wherein at least one antibody or antibody fragment furthercomprises a cell penetrating peptide and/or is an intrabody.
 46. Amethod of detecting a PD-L1 or PD-L2 expressing cell in a subjectcomprising: (a) contacting a sample from said subject with an antibodyor antibody fragment having a first set of clone paired heavy and lightCDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and a second setof clone paired heavy and light CDR sequences selected from SEQ ID NOS:31-36 or 37-42; and (b) detecting a PD-L1 or PD-L2 expressing cell insaid sample by binding said antibody or antibody fragment to a cell insaid sample.
 47. The method of claim 46, wherein said sample is a bodyfluid.
 48. The method of claim 46, wherein said sample is tissue sample.49. The method of claim 46, wherein detection comprises ELISA, RIA orWestern blot.
 50. The method of claim 46, further comprising performingsteps (a) and (b) a second time and determining a change in antigenlevels as compared to the first assay.
 51. The method of claim 46,wherein said antibody or antibody fragment is encoded by a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 3-4 or 5-6, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 7-8 or 9-10.
 52. Themethod of claim 46, wherein said antibody or antibody fragment isencoded by heavy and light chain variable sequences having at least 70%,80%, or 90% identity to a first set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 3-4 or 5-6, and asecond set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 7-8 or 9-10.
 53. The method of claim 46,wherein said antibody or antibody fragment is encoded by heavy and lightchain variable sequences having at least 95% identity to a first set ofclone paired heavy and light chain variable sequences selected from SEQID NOS: 3-4 or 5-6, and a second set of clone paired heavy and lightchain variable sequences selected from SEQ ID NOS: 7-8 or 9-10.
 54. Themethod of claim 46, wherein said antibody or antibody fragment comprisesa first set of clone paired heavy and light chain variable sequencesselected from SEQ ID NOS: 11-12 or 13-14, and a second set of clonepaired heavy and light chain variable sequences selected from SEQ IDNOS: 15-16 or 17-18.
 55. The method of claim 46, wherein said antibodyor antibody fragment comprises heavy and light chain variable sequenceshaving at least 70%, 80%, or 90% identity to a first set of clone pairedheavy and light chain variable sequences selected from SEQ ID NOS: 11-12or 13-14, and a second set of clone paired heavy and light chainvariable sequences selected from SEQ ID NOS: 15-16 or 17-18.
 56. Themethod of claim 46, wherein said antibody or antibody fragment comprisesheavy and light chain variable sequences having 95% identity to a firstset of clone paired heavy and light chain variable sequences selectedfrom SEQ ID NOS: 11-12 or 13-14, and a second set of clone paired heavyand light chain variable sequences selected from SEQ ID NOS: 15-16 or17-18.
 57. The method of claim 46, wherein the antibody fragment is arecombinant di-scFv (divalent single chain fragment variable) antibodyor F(ab′)₂ fragment.
 58. The method of claim 46, wherein said cell is acancer cell.
 59. The method of claim 58, wherein the cancer cell is alymphoma cell, a breast cancer cell, or renal cell carcinoma cell. 60.The method of claim 58, wherein said cell is a cell associated withimmune suppression.
 61. The method of claim 60, wherein said cellassociated with immune suppression is a non-cancerous cell in the tumormicroenvironment.
 62. The method of claim 61, wherein said non-cancerouscell in the tumor microenvironment is a stromal or endothelial cell. 63.A method of treating immune suppression in a tumor microenvironment in asubject having cancer comprising delivering to said subject an antibodyor antibody fragment having a first set of clone paired heavy and lightCDR sequences selected from SEQ ID NOS: 19-24 or 25-30, and a second setof clone paired heavy and light CDR sequences selected from SEQ ID NOS:31-36 or 37-42.